Novel pyrazoline and pyrazole “turn on” fluorescent sensors selective for Zn2+/Cd2+ at λem 480 nm and Fe3+/Fe2+ at λem 465 nm in MeCN

A small series of simple pyrazoline and pyrazole based sensors, all derived from the same chalcone precursors, were synthesised, characterised and screened for their fluorescence “turn on” properties in the presence of multiple metals. Pyrazole 8 displayed an excellent fluorescence profile with approx. 20× fold increase in λem 480 nm with Zn2+ compared to a 2.5× fold increase with Cd2+. Pyrazole 9 displayed a 30× fold increase at λem 465 nm for Fe3+ compared to Fe2+ with a Fe3+ limit of detection of 0.025 μM. The corresponding pyrazolines displayed contrasting properties with important implications for future pyrazoline and pyrazole sensor design.


Introduction
Zinc is the second most abundant transition metal in the human body 1 critical to a diverse range of biological functions including enzyme maintenance, 2 gene expression 3 and neurological functions. 4Unregulated zinc is implicated in a number of biological illnesses ranging from Alzheimer's disease, 5 epilepsy 6 and Parkinson's disease. 7Cadmium, also a group 12 transition metal sitting beneath zinc in the periodic table, is a highly toxic environmental and industrial pollutant with long term exposure linked to renal, breast and lung cancers. 8,91][12] Fluorescence sensing typically involves two common approaches, either a "turn on" sensor 13,14 in which the presence of the analyte of interest increases uorescence emission intensity (l em ) or a "turn off" sensor 15,16 in which the analyte decreases uorescence intensity.A major challenge remains which is the ability to selectively detect zinc over cadmium and vice versa in complex mixtures, with only a few examples having been reported in the literature. 17ultiple heterocyclic scaffolds are used to tune metal ion selection, photophysical and chemical properties to meet a particular sensor requirement.Pyrazoline, 18 a 5 membered heterocyclic ring, and the closely related pyrazole 19 are leading examples combining the advantageous properties of modular design from commercially available starting materials and the ability to Taylor the orientation of the three branching units off the main pyrazoline or pyrazole core (shown in blue and red respectively in Fig. 1).Pyrazolines chelators for gold, 20 tin 21 and ruthenium 22 have all been reported with the groups of Miao and Zhao pioneering both "turn on" and "turn off" pyrazoline uorescent sensors for multiple systems including Zn 2+ in aqueous environments and living systems. 23A variety of photophysical processes result in increased uorescence emission 18,19 with the blocking of the photoinduced electron transfer Fig. 1 Pyrazoline heterocycle (shown in blue) in fluorescent "turn on" and "turn off" sensors developed by Miao and Zhao groups. 23Inset demonstrates "turn on" fluorescent sensor pyrazoline A can be converted into pyrazole B with different photophysical properties. 25

PAPER
17a,23a,35 Mixed uorescent sensors composed of both pyrazoline and pyrazole units have also been reported 24 highlighting that structural complexity is not a prerequisite for complex functionality, simple molecular structures provide the tools to detect biologically important analytes in living systems.
Previous work 25 reported simple pyridine pyrazoline A as a "turn on" uorescent sensor for both Zn 2+ and Cd 2+ producing a 1.7× fold higher response for Cd 2+ /Zn 2+ (Fig. 1 inset).Oxidation of A to the corresponding pyrazole B produced a "turn on" sensor capable of distinguishing Zn 2+ /Cd 2+ with l em 380 nm and l em 350 nm respectively.Herein we report the next generation of sensors with three novel pyrazolines 4-6 and their related three novel pyrazoles 7-9 with pyrazole 8 able to detect Zn 2+ with a 20× fold increase in uorescence at l em 480 nm compared to absence of Zn 2+ .Cd 2+ resulted in only a 2.5× fold increase in uorescence at l em 480 nm.In contrast, pyrazole 9 displayed only a minor increase in uorescence with Zn 2+ and Cd 2+ but 30× fold increase in l em 465 nm with Fe 3+ but surprisingly not Fe 2+ .These results within provide valuable insight for the design of the next generation of pyrazoline and pyrazole uorescent sensors specic for Zn 2+ and Fe 3+ .
2 Results and discussion 2,6-Diacetylpyridine underwent Claisen-Schmidt condensation 26 with the required substituted aromatic aldehyde to afford the chalcone precursors 1-3 in acceptable yield (34-73%).Aer extensive investigation, it was discovered that a 2 : 1 ratio of ketone to aldehyde with catalytic amount of NaOH promoted formation of the required mono-chalcone and minimal formation of the bis-chalcone side product (ESI S3 †).Bis-chalcone could be easily removed by gravity ltration allowing the desired chalcone to be further puried by recrystallisation preventing the requirement for time consuming column chromatography.Conversion of chalcone into pyrazoline 4-6 was achieved (ESI S2 †) by adapting previous methods 25,27 involving the 1,2 addition of methylhydrazine in slight excessive at room temperature (36-43% yield).Hydrazone formation was possible however following an extensive aqueous wash only the desired product was observed, this is comparable with other reports of hydrazone hydrolysis under similar conditions in the literature (Scheme 1). 31his method was modied to yield the closely related pyrazole series 7-9 using excess methylhydrazine and longer reaction time to afford the pyrazole series in satisfactory yield (8-18%). Oxidation of pyrazoline to pyrazole has been reported previously 25,28 with only a few literature examples of direct transformation from chalcone to pyrazole 29,30 typically involving a catalyst and/or heating.One pot pyrazole synthesis from chalcone using excess methylhydrazine was conrmed by 1 H NMR spectroscopy with formation of an aromatic pyrazole 1 H singlet signal at approx.7.1 ppm (H d in Fig. 4 for 7, ESI S3 † for 6 and 9) with the absence of the three sets of doublet of doublet signals characteristic of a pyrazoline ring reported previously. 25,27High resolution mass spectrometry conrmed pyrazole formation.15][16][17][18][19][20]23,25 Zn 2+ chelation for the pyrazoline series was conrmed using UV/Vis spectroscopy in MeCN, this solvent was selected to enable direct comparison with previous studies 25 and to determine the optimum photophysical properties for a future watersoluble sensor.The initial absorbance band at 320 nm (3 = 16 550 M −1 cm −1 ) decreasing with the linear appearance of a new band at 340 nm up to 2.0 equivalents (eq.)Zn 2+ (Fig. 2 for pyrazoline 5 and ESI S4 † for 4 and 6).All three pyrazolines exhibited similar absorbance proles suggesting substitution at the aryl ring was not detrimental to chelation.
A 1 : 2 ratio of sensor to Zn 2+ was suggested from the linear increase in absorbance at 375 nm observed up to 2 eq. of Zn 2+ (Fig. 2 inset) with further increases producing no signicant increase in absorbance.Job plot analysis 32 (Fig. 3 for 5 ESI S5 † for 4-9) also indicated a 1 : 2 ratio between all pyrazolines and Zn 2+ .This is in contrast to pyrazoline A which had a 1 : 1 ratio of sensor to Zn 2+ .The addition of the acetyl group may be responsible for this additional Zn 2+ chelation.
Fluorescence spectroscopy was used to screen multiple metals to determine useful uorescence properties.Pyrazoline 4 remains more sensitive to Cd 2+ than Zn 2+ with a 7.3× fold increase at l em 480 nm compared to 4.5× fold with Zn 2+ (Fig. 5).It is interesting to note the similarity with the previous reported sensor A 25 lacking the acetyl group also displayed higher uorescence with Cd 2+ over Zn 2+ with a ratio of 1.7× fold higher emission at the same l em 465 nm with Cd 2+ over Zn 2+ , comparable to the 1.6× fold reported above.A Zn 2+ limit of detection (LoD) 33 of 0.0319 mM for 4 and 0.010 mM for 5 was calculated (see ESI S7 †) which is similar to the 0.0202 mM Zn 2+ LoD for A 25 other Zn 2+ based "turn on" uorescent sensors.23b This suggests the additional acetyl group is not conveying any benecial effect in terms of Zn 2+ /Cd 2+ analyte selectivity but provides a slight improvement in uorescence response.This should be factored into future pyrazoline based sensor design.It is noteworthy to highlight further chemical modication to introduce additional functional groups via the acetyl group may enhance "turn on" uorescence properties and this is ongoing research in our laboratory.
Previous studies demonstrated pyrazolines can be converted into the closely related pyrazoles displaying "turn on" uorescence properties in the presence of different cations.To investigate further, pyrazole series 7-9 were screened across multiple metals and to our surprise pyrazole 8 demonstrated an excellent "turn on" uorescence response for Zn 2+ /Cd 2+ (Fig. 6 for 8, ESI S6 † for 7).
Upon addition of 5 eq.Zn 2+ the uorescence at l em 480 nm increased 20× fold whereas the addition of 5 eq.Cd 2+ only resulted in a 2.5× fold increase at l em 480 mn resulting in approximately 8× fold increase in selectivity for Zn 2+ over Cd 2+ .This is in contrast to the previously reported pyrazole B 25 lacking the acetyl group which displayed a 13× fold increase l em 380 nm upon addition of 5 eq.Zn 2+ .Job plot analysis (ESI S5 †) suggests a 1 : 1 ratio between 8 and Zn 2+ similar to pyrazole B reported previously. 25Pyrazole 7 did not display a signicant increase in uorescence on addition of Zn 2+ or Cd 2+ (see ESI S6 †) suggesting the electronegative uorine group on the aryl ring was a key requirement of this "turn on" uorescent response.Substitution of the 4-F for an electron donating 4-OMe group resulted in 9 which abolished this Zn 2+ response.investigation revealed 9 displayed a 30× fold increase at l em 465 mn with Fe 3+ but not Fe 2+ (Fig. 7).
The difference was profound and could be visibly observed using a low power 6 W l ex 254 nm TLC lamp (Fig. 7 inset).Job plot analysis (ESI S5 †) suggested a 1 : 2 sensor to Fe 3+ ratio.Pyrazole 9 is an excellent candidate for a "turn on" uorescent sensor with a calculated Fe 3+ LoD of 0.025 mM.This is noteworthy as the rst reported Fe 3+ specic pyrazoline sensor had a Fe 3+ LoD of 3 mM (ref.34) with recent pyrazole based Fe 3+ sensors reporting Fe 3+ LoD ranging from 0.021 mM (ref.35) to 0.0004 mM. 24The proposes 1 : 1 binding mechanism of pyrazole 8 with Zn 2+ and a 1 : 2 binding mechanism of pyrazole 9 with Fe 3+ is displayed in Fig. 8.][38] Competition assays were performed to assess pyrazole 8 Zn 2+ "turn on" uorescence response in the presence of competing metal cations (Fig. 9).Fluorescence quenching was observed upon addition of a range of paramagnetic metals including Fe 3+ , Ni 2+ and Co 2+ , a common phenomenon observed in the literature. 25A good uorescence response in the presence of Na + and K + cations was observed suggesting the presence of these singularly charged metals was not detrimental to the Zn 2+ sensing properties of 8.
Pyrazole 9 was also submitted to a competition assay to evaluate its ability as a Fe 3+ "turn on" sensor and it displayed a slightly better prole than 8 retaining modest uorescence response in the presence of Fe 2+ , Mn 2+ , Cu 2+ , Ru 3+ and Co 2+ (Fig. 10).Unfortunately, the presence of Na + and K + did significantly reduce l em 455 nm uorescence in contrast to 8.
The competition assay proles for 8 and 9 are similar to previously reported pyrazolines with paramagnetic metals typically hampering uorescent response. 25Further work is required to enhance analyte chelation and prevent competing cations from disrupting the "turn on" response.The unexpected switch from a Zn 2+ to a Fe 3+ "turn on" sensor for 8 and 9 highlight the modular nature of the pyrazole heterocycle and how small modications can have a profound inuence on photophysical properties.

Conclusions
The addition of an acetyl group on the pyridine of pyrazole sensor 8 improved uorescence properties (20× fold increase at l em 480 nm) for the detection of Zn 2+ over Cd 2+ (2.5× fold increase also at l em 480 nm) compared to sensor B 25 reported previously.Substitution of the electronegative 4-F on pyrazole 8 for an electron donating 4-OMe group in 9 resulted in the unexpected discovery of a "turn on" uorescent sensor for Fe 3+ at l em 465 nm. 9 displayed a Fe 3+ LoD of 0.025 mM which is comparable to recently reported Fe 3+ uorescent sensors. 24,35g. 7 Fluorescence spectra of pyrazoline 9 on addition of 5 eq. of the indicated metal.Inset from left to right, +Fe 3+ , 9 only, +Fe 2+ with l ex 254 nm 6 W lamp. 100 mM 9 with 500 mM metal cations.These results suggest the acetyl group highly benecial and should be factored into future pyrazole sensor design.In contrast the same modication to pyrazoline sensors conferred no signicant advantage in selectivity towards Zn 2+ /Cd 2+ compared to sensor A 25 however it did produce a slight increase in Zn 2+ LoD.The above studies were performed in MeCN to aid comparison to previous work which was also carried out in MeCN.Previous exploratory reports for Zn 2+ uorescent sensors were performed in pure organic solvents also including MeOH, 39,40 THF, 41,42 DMF 43 and DMSO. 44,45The results within provide a rm foundation for developing aqueous based sensors for lead pyrazole 8 with Zn 2+ and 9 with Fe 3+ , this is ongoing work and will be reported in due course.

Fig. 10
Fig. 10 Competition experiments for pyrazole 9.The white bar represents 9 (MeCN, 20 mM, l ex 290 nm, l em = 455 nm) with 5 eq. of the indicated cation; the black bars is the same plus 5 eq.Fe 3+ after equilibrating for 3 min.